Novel epidermis/dermis equivalents and aged skin equivalents shaped therefrom

ABSTRACT

An aged skin equivalent comprising an epidermis equivalent and an aged dermis equivalent, wherein the aged dermis equivalent comprises glycated collagen.  
     An aged dermis equivalent, the epidermis equivalent obtained and methods of producing the aged skin and/or aged dermis equivalent and/or the epidermis equivalent.

CROSS-REFERENCE TO PRIORITY APPLICATION

[0001] This application claims priority under 35 U.S.C. §119 ofFR-99/04970, filed Apr. 20, 1999, hereby expressly incorporated byreference.

BACKGROUND OF THE INVENTION

[0002] 1. Technical Field of the Invention

[0003] The present invention relates to novel skin equivalents, to amethod for the production thereof and to the epidermis equivalents anddermis equivalents comprising such novel skin equivalents (themselvesper se novel).

[0004] 2. Description of the Prior Art

[0005] For several years, the perfection of models of reconstructed skinwhich permit conducting studies required for a better understanding ofthe role of the skin in the mechanical domain and physiological domainhas been ongoing.

[0006] Thus, models which more or less approximate human skin have beendeveloped. Exemplary are the models described in EP-A-285471,EP-A-285474, EP-A-789074, EP-A-502172, EP-A-418035, WO-A-9116010,EP-A-197090, EP-A-20753, FR-A-2665175 and FR-A-2689904.

[0007] Very generally, the models of reconstructed skin described in theaforesaid publications comprise human keratinocytes associated orotherwise with other skin cells such as melanocytes and/or Langerhanscells, deposited onto a support, often a dermis equivalent, and culturedunder conditions such that they commence a program of differentiationwhich results in the formation of an epidermis equivalent.

[0008] The dermis equivalents described to date are either artificialmembranes such as, for example, Millipore filters, collagen-basedsubcutaneous substitutes, plastic or any other support which iscompatible with cell viability, or supports which are more developedsuch as to approximate natural dermis, for example previouslyde-epidermilized dermis or Collagen/fibroblast mixed lattices.

[0009] In the collagen/fibroblast mixed lattices the association ofnative collagen and isolated human fibroblasts ultimately provides adermis equivalent which mimics a dermis which has not been subjected tothe actions of the weather.

[0010] The protocols employed for the preparation of said lattices usecollagen originating in general from young tissues, this collagenhaving, however, been subjected to the important post-translationalmodifications which intervene in the complex but nevertheless normalprocesses of its biosynthesis, but not having undergone all of themodifications which can intervene, in particular, during aging.

[0011] It is known to this art, in particular, to this art, that duringaging and during the progress of certain diseases such as diabetes,non-enzymatic processes operate which involve an ose (glucose or ribose)which reacts according to the Maillard reaction with an amino group (forexample a lysine residue) of the collagen to form a Schiff's base. Thisbase, after undergoing an Amadori molecular rearrangement, may provide,by a succession of reactions, intramolecular bridging such as, forexample, of pentosidine type. This phenomenon, termed glycation ofcollagen, increases uniformly with age, leading to a uniform increase inthe glycation-product content of the skin. These glycation productsinclude, for example, pyrraline, carboxymethyl-lysine, pentosidine,crosslines, N^(ε)-(2-carboxyethyl)lysine (CEL), glyoxal-lysine dimer(GOLD), methylglyoxal-lysine dimer (MOLD), 3DG-ARG imidazolone,versperlysines A, B, C, threosidine or, alternatively, advancedglycosylation end products or AGEs. This phenomenon is amplified incertain disease states, such as, for example, diabetes.

[0012] Without wishing to be bound to any particular theory as regardsaging of the skin, it should be noted that other characteristics whichmight also be a consequence of these glycation phenomena, such as adecrease in heat denaturation, an-increase in resistance to enzymaticdigestion and an increase in intermolecular bridging, have beendemonstrated during aging of the skin (Tanaka S. et al., 1988, J. Mol.Biol., 203, 495-505; Takahashi M. et al., 1995, Analytical Biochemistry,232, 158-162). In addition, modifications due to the glycation ofcertain constituents of the basal membrane such as collagen IV, laminineand fibronectin have been demonstrated (Tarsio J F. et al., 1985,Diabetes, 34, 477-484; Tarsio J F. et al., 1988, Diabetes, 37, 532-539;Sternberg M. et al., 1995, C. R. Soc. Biol., 189, 967-985).

[0013] Thus, it is understood that during aging of the skin thephysicochemical properties of collagen are modified and such collagenbecomes more difficult to dissolve and more difficult to degrade.

[0014] Thus, one of the components of aged skin indeed appears to beglycated collagen.

[0015] It is also very well known that the skin constitutes a closeassociation between at least two components thereof, i.e., the epidermisand the dermis. The interactions between the dermis and the eipdermisare such that it is reasonable to consider that a modification of onemay have consequences on the other. It may be suspected that the agingof the dermis, in particular with its glycation phenomena, can only haveconsequences for the epidermis to which it is associated. Thus, duringskin aging, the glycation of the collagen must promote modifications ofthe epidermis which must participate in the aging of the epidermis.

[0016] In this respect, it has now been demonstrated that a constitutiveprotein of normal epidermis, i.e., the β1 type integrin, anextracellular matrix receptor (see Ruoslahti E., 1991, Cell Bioloay ofExtracellular Matrix, Plenum press New York, 343363), shows adistribution of its expression in aged epidermis which is very differentfrom that in young epidermis. Specifically, while in a young epidermis,i.e., for the purposes of this invention an epidermis from a youngsubject, this protein is expressed-in the very deep layers of theepidermis, i.e., up to a maximum of the second suprabasal layer, thesituation is completely different in an aged epidermis, i.e., for thepurposes of this invention in an epidermis from an elderly subject,where this protein is expressed in most layers of the epidermis,directly through to the last suprabasal layers under the cornifiedlayer.

[0017] To date, no model of reconstructed skin in vitro is capable,either because of the protocols of preparation technique, or the simplefact that once reconstituted it does not undergo modification, ofproducing a skin equivalent at least one of the constituents of whichprovides one of the components of skin aging. Thus, no model ofreconstructed skin in vitro presents the properties of an aged skin orpermits the study of the processes resulting therein, or the study ofthe compounds and/or compositions which would at least make it possibleto slow down or retard this/these process(es). The only knownevaluations of these phenomena entail in vivo studies, either in animalsor humans. Most particularly, for ethical reasons, the advantage ofhaving such a model is conspicuously apparent.

[0018] Studies on glycation are known in the prior art. For example, amethod is described for obtaining a connective tissue equivalent in theform of a lattice of glycated collagen and fibroblasts (see in thisrespect Frey et al. (1992, C. R. Soc. Biol., 187, 223-231). However, notonly have Frey et al. never investigated or even suggested thepossibility of preparing a skin equivalent from their lattice, they havenever compared this lattice to any dermis equivalent. In addition, butwhile recognizing the validity of the conjunctive tissue model of Freyet al., it will be appreciated that the protocol employed cannot providethe objectives established hereby, i.e., to reproduce in vitro a skin,and consequently an epidermis and a dermis, which has all or part of theproperties of an aged skin.

[0019] By incubating the collagen and sugar for 9 hours at a temperatureof 4° C., Frey et al. initiate the collagen glycation reaction, thisreaction then continuing in the lattice in which the collagen is in thismanner preglycated. If it is desired to attain a sufficient level ofglycation, such as to mimic an aged skin, it is then necessary to permitthe glycation reaction to continue in the lattice formed in thisfashion, i.e., in the process of contraction, for a further time whichis sufficient to attain the desired level. However, those skilled inthis art are cognizant that to establish an epidermis equivalentcontaining at least keratinocytes on a dermis equivalent ofcollagen/fibroblast lattice type, the seeding of the keratinocytes mustbe conducted onto a lattice which cannot have exceeded a determinedstage of contraction. It will thus be seen, therefore, that it is notpossible to obtain by the Frey protocol a dermis equivalent which mimicsan aged or very aged dermis, i.e., highly or very highly glycated.

SUMMARY OF THE INVENTION

[0020] Accordingly, a major object of the present invention is theprovision of a skin equivalent which comprises an epidermis equivalentproduced on an aged dermis equivalent, particularly on-a latticecomprising at least glycated collagen and fibroblasts.

[0021] A significant advantage of this invention is providing a model ofreconstructed skin in which at least one of the components thereof wouldhave one of the aspects of an aged skin.

DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED EMBODIMENTS OFTHE INVENTION

[0022] More particularly according to the present invention, theassignee hereof has long been interested in the production of in vitromodels of reconstructed skin, and a novel model of reconstructed skinhas now been developed comprising an epidermis equivalent and a dermisequivalent, said dermis equivalent comprising an aged dermis equivalent,particularly a lattice which comprises at least glycated collagen andfibroblasts.

[0023] Thus, the present invention features a novel aged dermisequivalent, comprising at least glycated collagen and fibroblasts.

[0024] Different techniques for tracking the formation of the glycationproducts are described in the prior art. In this respect, methods whichare representative are described by Cefalu W. T. et al. (Journal ofGerontoloay; Biological Sciences, 1995, vol. 50 A, No. 6, 13337-13341),by Sell D. R. (Diabetes/Metabolism, 1991, vol. 7, No. 4, 239-251), byMiyata T. et al. (Journal of the American Society of Nephroloay, 1996,vol. 7, No. 8, 1198-1206), or, alternatively, by Furth A. J. (AnalyticalBiochemistry, 1991, 192, 109-111).

[0025] Thus, it is possible to measure the level of glycating compoundbound to the collagen and/or the level of glycating compound remainingafter the reaction. As described above, the glycation of collagen leadsto the formation of glycation products such as, for example, pyrraline,carboxymethyl-lysine, pentosidine, crosslines,N^(ε)-(2-carboxyethyl)lysine (CEL), glyoxal-lysine dimer (GOLD),methylglyoxal-lysine dimer (MOLD), 3DG-ARG imidazolone, versperlysinesA, B and C, threosidine or alternatively advanced glycosylation endproducts or AGEs.

[0026] Certain of these glycation products have the particular featureof emitting a measurable fluorescence after excitation. For example,pentosidine, when excited at a wavelength (λex) of 328 nm, emits afluorescence at a wavelength (λem) of 378 nm. Similarly, AGEs, whenexcited at a wavelength (λex) of 370 nm, emit a fluorescence at awavelength (λem) of 440 nm. The ratio of the fluorescence which isemitted by a given glycation product in the dermis equivalent comprisingat least glycated collagen and fibroblasts to the fluorescence which isemitted by the same glycation product in a control dermis equivalentcomprising at least non-glycated collagen and fibroblasts, measuredunder the same experimental conditions, makes it possible tocharacterize the level of glycation of the aged dermis equivalent.

[0027] Particularly, according to the invention, the fluorescence of thepentosidine and/or the AGEs is measured both in the aged dermisequivalent comprising. glycated collagen and in a control comprisingnon-glycated collagen.

[0028] Thus, according to the invention, the aged dermis equivalent hasa level of glycation ranging from 2 to 30 and particularly from 8 to 18.

[0029] Consistent herewith, the-collagen may be any type of collagen ofany origin. In this respect, reference is made to the different types ofcollagen noted in the reviews of Van der Rest and Garonne, 1990,Biochem., vol. 72, 473-484 or, alternatively, 1991, Faseb Journal, vol.5, 2814-2823. Thus, according to the invention, the collagen ispreferably selected from among the fibrillary collagens of type I, IIIor V.

[0030] Preferably, collagen of animal origin is used, particularlycollagen of bovine origin.

[0031] The preferred collagen according to the invention is type Icollagen. Very preferably, type I bovine collagen is employed.

[0032] It will of course be appreciated that a mixture of collagen ofdifferent types in any proportion and/or of different origins may beused.

[0033] Also according to the invention, the fibroblasts may emanate fromany origin, but are preferably fibroblasts of human origin. They may beprepared according to any known technique of the prior art, for exampleby mechanical and/or enzymatic dissociation of the macromolecules of theextracellular matrix of the dermis or by growth of fibroblasts fromexplants.

[0034] Too, the dermis equivalent of the invention comprises at leastglycated collagen and fibroblasts, but may also contain any othercomponent that might be advantageous to introduce therein such as, forexample, endothelial cells, macrophages or alternatively nerve cells.

[0035] The present invention also features an epidermis equivalentcomprising at least keratinocytes, characterized in that it can beformed by seeding at least keratinocytes onto a dermis equivalentcomprising at least glycated collagen and fibroblasts.

[0036] As above indicated, depending on age, certain markers of theeipdermis could undergo modifications either in their quantity or in thelocalization of their expression. Particularly, it has been shown thatthe expression of β1 integrin in the epidermis is modified in itslocalization with the age of the epidermis. Specifically, while in ayoung epidermis this protein is expressed strictly in a maximum of thefirst two suprabasal layers, with age this expression, while conservingits localization in the first two suprabasal layers, appears in layerswhich are more and more superficial to the point of appearing in all ofthe suprabasal layers including the final layers adjoining the stratumcorneum.

[0037] Thus, in one embodiment of the invention the epidermis equivalentis characterized in that it has modified expression of β1 integrin,particularly expression of β1 integrin in the cells of at least thefirst three suprabasal layers.

[0038] Very preferably, according to the invention, the epidermisequivalent comprising at least keratinocytes is characterized in that ithas modified expression of β1 integrin, particularly expression of β1integrin in the cells of at least the first three suprabasal layers, andin that it is obtained by seeding at least keratinocytes onto a dermisequivalent comprising at least glycated collagen and fibroblasts.

[0039] Of course, any method which makes it possible to demonstrate theexpression of β1 integrin can be used to characterize the epidermis ofthe invention. Labelling with the aid of anti-β1 integrin antibodies isexemplary.

[0040] The keratinocytes used according to the invention may emanatefrom any origin, but are preferably keratinocytes of human origin. Theymay be prepared according to any known method of the prior art.Culturing from dissociated epidermis originating from a normal skinsample, or the culturing of keratinocytes derived from the sheath of thehair follicles, are representative examples.

[0041] Preferably, keratinocytes from normal human skin are used.

[0042] Also, preferably, the keratinocytes are prepared from dissociatedhuman epidermis originating from a normal human skin sample according tothe technique described in Régnier et al., Frontier of Matrix Bioloay,Vol. 9, 4-35 (Karger, Basle 1981).

[0043] The epidermis equivalent of the invention comprises at leastkeratinocytes, but it may comprise any other cell type which might beincorporated therein such as, for example, Langerhans cells and/orprecursors of Langerhans cells and/or melanocytes.

[0044] Of course, the skin equivalent which has the best similarity withnormal skin is the skin equivalent which contains the three essentialcell types present in normal skin.

[0045] Thus, advantageously the model of reconstructed skin according tothe invention comprises, in addition, melanocytes and/or Langerhanscells and/or precursors of Langerhans cells.

[0046] The melanocytes according to the invention may be isolated fromany organ which contains them such as, for example, normal skin or hairfollicles.

[0047] Preferably, melanocytes isolated from normal skin are used.

[0048] Any known method of preparation of melanocytes of the prior artmay be used according to the invention. The method described in Olssonet al., Acta Derm. Venereol., 1994, 74, 226-268, is one such example.

[0049] The Langerhans cells and/or the precursors of Langerhans cellswhich can be employed according to the invention may be as described inEuropean Patent Application published under the number EP-A-789074,assigned to the assignee hereof.

[0050] This invention also features an aged skin equivalent, whichcomprises at least one epidermis equivalent and one aged dermisequivalent.

[0051] In another embodiment of the invention, the aged skin equivalentcomprises an aged dermis equivalent as described above.

[0052] In another embodiment of the invention, the aged skin equivalentcomprises an epidermis equivalent as described above.

[0053] An aged skin equivalent which is very preferable according to theinvention comprises an epidermis equivalent comprising at leastkeratinocytes which have modified expression of β1 integrin,particularly expression of β1 integrin in the cells of at least thefirst three suprabasal layers and is characterized in that it can beobtained by seeding at least keratinocytes onto an aged dermisequivalent comprising at least glycated collagen and fibroblasts, saidaged dermis equivalent having a level of glycation ranging from 2 to 30,and particularly from 8 to 18.

[0054] The present invention also features a method for preparing anaged skin equivalent comprising an epidermis equivalent and a dermisequivalent which itself comprises a lattice comprising at least glycatedcollagen and fibroblasts, wherein a first step a lattice comprising atleast glycated collagen and fibroblast is prepared and in a second stepan epidermis equivalent comprising at least keratinocytes isreconstituted on the lattice obtained in the first step.

[0055] The first step may be carried out by any known technique of theprior art, provided that the collagen may be glycated either beforehand,during or after the formation of the lattice. Preferably, according tothe invention, a lattice is prepared in which either collagen which isglycated prior to the formation of the lattice is used or a glycationagent is added to the mixture of collagen and fibroblast which is used,in order to effect the glycation either during the preparation of thelattice or after formation of the lattice.

[0056] Preferably, the lattice is prepared using previously glycatedcollagen.

[0057] Also preferably, the lattice is prepared according to the methoddescribed by Asselineau et al., 1987, (Models in Dermato., vol. III, Ed.Lowe & Maibach, 1-7) using preglycated collagen.

[0058] Any known method of glycation may be used to produce glycatedcollagen. For example, the methods described by Tanaka et al. (J. Mol.Biol., 1988, 203, 495-505), Tarsio J F. et al. (1985, Diabetes, 34,477-484), Tarsio J F. et al. (1988, Diabetes, 37, 532-539) or,alternatively, by Frey J. et al. (1992, C. R. Soc. Biol., 187, 223-231)are exemplary thereof.

[0059] Preferably, the glycation may be carried out by contacting asolution of at least one collagen with a solution of at least oneglycating agent in such manner as to induce the glycation reaction ofthe collagen in vitro in the absence of cells.

[0060] As indicated above, the collagen may be any type of collagen, ofany origin, alone or in admixture.

[0061] Preferably, the collagen is of animal origin, particularlycollagen of bovine origin.

[0062] The preferred collagen according to the invention is type Icollagen. Very preferably, type I bovine collagen is employed.

[0063] The collagen solution is advantageously at a concentration offrom 2 mg/ml to 6 mg/ml and preferably from 3 mg/ml to 5 mg/ml.

[0064] The glycating agent may be any agent which enables the glycation,i.e., which is capable of reacting according to the Maillard reactionwith an amino group of the collagen to form a Schiff's base. In thisrespect, exemplary intermediates of the Maillard reaction include forexample, glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal or,alternatively, sugars.

[0065] Any type of sugar can be used according to the invention, whetherit is in monomeric or polymeric form. According to the invention, amonomeric sugar is preferred.

[0066] By “sugar” are intended compounds which possess several alcoholgroups and at least one aldehyde group. Oses are particularlyrepresentative thereof.

[0067] Among the sugars which are suitable according to the invention,ribose, fructose or glucose, inter alia, are exemplary. Preferably,ribose or glucose is used.

[0068] The sugar may be in any one of the dextrorotatory orlaevorotatory conformations. Preferably, a sugar in dextrorotatoryconformation is used.

[0069] Particularly, according to the invention D-fructose, D-ribose orD-glucose is used. Preferably D-ribose or D-glucose is used.

[0070] The glycating agent may be used either alone or in admixture.

[0071] The amount of glycating agent which can be used according to theinvention should be sufficient to permit initiating the non-enzymaticreactions which lead to the formation of Schiff's base. It should beunderstood that varying this amount makes it possible to obtain a finalproduct, the glycated collagen, whose level of glycation varies fromrelatively unglycated to very highly glycated. Thus, the amount ofglycating agent advantageously ranges from 0.5% to 20%, preferably from1% to 10% by weight of the total weight of the collagen solution.

[0072] The glycation reaction is carried out at a temperature which isclose to room temperature. Thus, the reaction is carried out at atemperature ranging from 15° C. to 30° C., preferably from 20° C. to 25°C.

[0073] The duration of the glycation reaction depends on the desiredlevel of glycation. It will be appreciated that the longer the time thehigher the level of glycation. Thus, the duration of the glycationreaction advantageously ranges from 15 days to 2 months, preferably from25 days to 35 days.

[0074] When it is determined to carry out the glycation prior to thepreparation of the lattice, it is possible to subject the glycatedcollagen to all the subsequent steps necessary for producing the purestpossible product. Thus, it is possible to attempt to eliminate any traceof glycating agent which would not have reacted during the reaction. Forthis any known technique may be employed. For example, the preglycatedcollagen solution is subjected to a series of dialyses against waterand/or acetic acid.

[0075] In another embodiment of the invention, the “preglycated”collagen solution obtained may be mixed with native collagen before usefor the preparation of the dermis equivalent. In this instance, theratio of glycated collagen to non-glycated collagen may range from 25 to75 and preferably from 45 to 55. Variation of this ratio makes itpossible to modulate the level of glycation of the lattice. Preferably,according to the invention a mixture of glycated collagen andnon-glycated collagen is used, and even more preferably a mixture ofglycated collagen and non-glycated collagen in a 50/50 ratio.

[0076] The second step of the method of the invention may be carried outby any known technique of the prior art.

[0077] In this respect, techniques which are representative aredescribed in EP-A-285471, EP-A-285474, EP-A-789074, EP-A-502172,EP-A-418035, WO-A-9116010, EP-A-197090, EP-A-20753, FR-A-2665175 andFR-A-2689904, or, alternatively, that described by Asselineau et al.,1985, (Exp. Cel. Res., 536-539) and 1987, (Models in Dermato., vol. III,Ed. Lowe & Maibach, 1-7).

[0078] Preferably, the method described by Asselineau and colleague isemployed.

[0079] The keratinocytes according to the invention may emanate from anyorigin, but are preferably keratinocytes of human origin. They may beprepared according to any known procedure of the prior art. Culture fromdissociated epidermis originating from a normal skin sample, or theculture of keratinocytes derived from the sheath of hair follicles, arerepresentative.

[0080] Preferably, the keratinocytes are prepared from dissociated humanepidermis originating from a normal human skin sample according to themethod described in Regnier et al., Frontier of Matrix Biology, Vol. 9,4-35 (Karger, Basle 1981).

[0081] Advantageously, after seeding the keratinocytes onto the support,the culture may be maintained submerged in a nutritive medium which maybe, for example, the medium described by Rheinwald and Green, 1975,(Cell, 6, (3), 317-330), this medium allowing the proliferation of thekeratinocytes (referred to hereinafter as medium 3F).

[0082] After an incubation time of 3 to 15 days, preferably of 7 to 9days, the skin equivalent is maintained at the air/liquid interface by,for example, depositing it onto a metal grid. The liquid then preferablyconsists of the same nutritive medium as the previous one.

[0083] The incubation then continues until production of a skinequivalent having the properties of a skin, i.e., the support on whichis an epidermis equivalent having the four conventional types of celllayer, i.e., the basal, suprabasal, granular and cornified layers.

[0084] Thus, the incubation continues for a duration advantageouslyranging from 5 days to 30 days, preferably from 7 days to 10 days.

[0085] The model of reconstructed skin produced in this manner comprisestwo entities: the support and the epidermis equivalent, that it ispossible to physically separate from each other.

[0086] The epidermis equivalent may thus be used separately from thesupport.

[0087] As above indicated, to date no in vitro model of reconstructedskin had the properties of an aged skin or permitted the study of theprocesses resulting therein or the study of the compounds and/orcompositions which would at least make it possible to modify the processthereof. The skin equivalent obtained according to the inventionresolves these problems, since it has at least one of the properties ofaged skin, i.e., a glycated collagen. This important property which themodel and method thereof make it possible to vary in any proportion,permits the study of the glycation phenomenon in itself and modulators(inhibitors or activators) of this phenomenon, the study of phenomenalinked to aged skin such as, for example, wrinkles, the study ofmodulators (isolated compounds and/or compositions) of the appearance ofthe wrinkles (particularly the inhibitors), the study of photoaging andthe effect on the skin of ultraviolet rays, as well as modulators ofthese effects (protective compounds and/or compositions, filters, etc.),the study of the influence of glycation on the components of the skin(bristles and/or hair, blood vessels, nerve fibers, etc.), and in thetherapeutic domain the study of the complications caused by diabetes viaglycation.

[0088] Thus, this invention also features employing an aged skin and/oraged dermis equivalent and/or an aged epidermis equivalent as describedabove for the study of the glycation phenomenon itself and modulators(inhibitors or activators) of this phenomenon, the study of phenomenalinked to aged skin such as, for example, wrinkles, the study ofmodulators (isolated compounds and/or compositions) of the appearance ofthe wrinkles (particularly the inhibitors), the study of photoaging andthe effect on the skin of ultraviolet rays as well as modulators ofthese effects (protective compounds and/or compositions, screeningagents, etc.), the study of the influence of glycation on the componentsof the skin (bristles and/or hair, blood vessels, nerve fibers, etc.),and in the therapeutic domain the study of the complications caused bydiabetes via glycation.

[0089] It will also been seen that, according to the invention, theepidermis equivalent which is reconstructed on the dermis equivalentcomprising at least glycated collagen and fibroblasts may have amodified distribution of the expression of β1 integrin. In thisinstance, if the variation in the distribution of this marker is linkedto the age of the epidermis, it is conceivable to use the aged skinequivalent of the invention to evaluate any product able to treat agedskin by measuring its effect by the effect that it produces on themodification of the distribution of β1 integrin expression in theepidermis equivalent.

[0090]FIG. 1 better illustrates the invention, without however limitingits scope. In this figure, the photographs show sections of skinequivalents after immunolabelling with the aid of an anti-β1 integrinantibody. Photo 1 represents the immunolabelling of a skin equivalentcomprising an epidermis equivalent reconstructed on a dermis equivalentwhich is prepared with non-glycated collagen. Photo 2 represents theimmunolabelling of a skin equivalent comprising an epidermis equivalentreconstructed on a dermis equivalent which is prepared with glycatedcollagen according to the invention.

[0091] In order to further illustrate the present invention and theadvantages thereof, the following specific examples are given, it beingunderstood that same are intended only as illustrative and in nowiselimitative.

EXAMPLES

[0092] Media and buffers unless otherwise indicated; all media andbuffers used in the following examples are described in Bell et al.,1979, (P.N.A.S. USA, 76, 1274-1278), Asselineau and Prunieras, 1984,(British J. of Derm., 111, 219-222) or Asselineau et al., 1987, (Modelsin Dermato., vol. III, Ed. Lowe & Maibach, 1-7).

Example 1 Preparation of Alycated Bovine Collagen I

[0093] 10 ml of bovine collagen I solution at a concentration of 3mg/ml, 0.8 ml of 0.5N sodium hydroxide to neutralize the acidic collagensolution and 100 μl of a 1M solution of D-ribose in water were placedinto a 50 ml Falcon tube.

[0094] The tube, rendered opaque to the light, was placed horizontallyand gently shaken at room temperature (25° C.) for 1 month.

[0095] At the end of the “preglycation”, the solution was placed indialysis tubing (Spectra/Poly labo 32 mm No. 132655/85716) and wassubjected to a series of successive dialyses:

[0096] 24 hours against demineralized water at 4° C. to eliminate theunbound sugar or the collagen degradation products;

[0097] 7 days against 0.5N acetic acid to redissolve the collagen, in 2baths of three and a half days;

[0098] 3×24 h against 0.017N acetic acid with bath changing every day.

[0099] After the last dialysis, the contents of the dialysis tubing wererecovered in a sterile beaker and the solution was transferred into asterile 50 ml Falcon tube, rendered opaque to the light.

[0100] The “preglycated” collagen solution was then ready to use. It maybe stored at 4° C.

Example 2 Preparation of an Aged Dermis Equivalent

[0101] 3.22 ml of 1.76×MEM medium, 0.63 ml of foetal calf serum, 0.35 mlof 0.1N sodium hydroxide and 0.20 ml of an MEM medium/Hepes mixturecontaining 10% foetal calf serum (MEM/Hepes/FCS10) were placed into asterile Falcon tube.

[0102] 0.50 ml of MEM medium/Hepes/FCS10 were then added containingfibroblasts derived from human mammary plastic surgery which wereprepared beforehand according to the technique described by Bell et al.,1979, (P.N.A.S. USA, 76, 1274-1278), Asselineau and Prunieras, 1984,(British J. of Derm., 111, 219-222) or Asselineau et al., 1987, (Modelsin Dermato., vol. III, Ed. Lowe & Maibach, 1-7), at a concentration of1×10⁶ cells for 0.5 ml of culture medium.

[0103] 2 ml of a volume/volume mixture of preglycated collagen fromExample 1 and non-glycated collagen having served in the preparation ofthe preglycated collagen from Example 1, at a concentration of 3 mg/mlin acetic acid at 1/1000, was then slowly added, against the wall of thetube, in such manner as to observe the appearance of a whiteish cloud.The entire medium was then mixed cautiously and plated in a 60 mmdiameter Petri dish (type Falcon 60 mm, ref. 1016). The Petri dish wasthen placed in an incubator at 37° C. and left for about 2 hours, 30minutes. When the appearance of 2 phases (gel+medium) was observed, thelattice was cautiously dissociated from its support and the latticedissociated from its support in this manner was left in the incubatorfor 4 days.

Example 3 Measurement of the Level of Glycation of the Aged DermisEquivalent From Example 2

[0104] In parallel to the production of the aged dermis equivalent ofExample 2, a dermis equivalent without glycated collagen (but withnon-glycated collagen) was produced. This equivalent was used as acontrol in the determination of the level of glycation of the glycateddermis equivalent from Example 2.

[0105] Two lattices (one aged, one control) prepared according toExample 2 were rinsed three times in phosphate buffered saline (PBS),then dried. The lattices were then placed in an Eppendorf tube andsubjected to digestion with pepsin (Sigma P-6887) at 37° C. in a waterbath overnight (12 hours) in a proportion of 500 μg of pepsin perlattice in 0.5 ml of 0.5N acetic acid.

[0106] 515 μl of 0.5N sodium hydroxide were then added to each tube andthe contents of each tube were filtered through a 0.22 μm spin filter(Sigma).

[0107] The fluorescence was then measured with the aid of a Hitachispectrofluorimeter, model F2000. The fluorescence emitted by pentosidineat λem=378 nm after excitation at λex=328 nm and the fluorescenceemitted by AGEs at λem=440 nm after excitation at λex=370 nm were thusmeasured.

[0108] The results obtained are reported in the Table below: TABLEPentosidine AGEs Control 650 430 Glycated lattice 6100 1820

[0109] In this example the level of glycation of the aged dermisequivalent was thus established at 9.4 for the pentosidine and at 4 forthe AGEs.

Example 4 Preparation of an Aged Skin Equivalent

[0110] An aged dermis equivalent as prepared in Example 2 was set up andwell spread out in a Corning θ 60 mm culture dish on a droplet ofcollagen “glue” (0.6 ml), then maintained at 37° C. in an incubator for20-30 minutes.

[0111] A sterile steel ring was placed on the lattice and 0.5 ml of acellular suspension of human keratinocytes originating from mammaryplastic surgery which were prepared according to Régnier et al.,(Frontier of Matrix Biology, Vol. 9, 4-35, Karger, Basle 1981), in aproportion of 100,000 cells/ml in MEM medium 10% FCS+3F, were placedinside the ring. About 6 ml of medium (MEM 10% FCS+3F) were placedaround the ring and the dish was placed in an incubator at 37° C. for 2hours. The ring was then removed and the dish again placed in theincubator.

[0112] After 8 days, the culture was then placed at the air/liquidinterface, said liquid consisting of the same medium as above.

[0113] The culturing was then continued for 1 week until production ofan epidermis equivalent which was histologically satisfactory, i.e., anepidermis equivalent which had the four conventional cell layers, i.e.,the basal, suprabasal, granular and cornified layers.

Example 5 Characterization of the Expression of β1 Intearin in theEpidermis Equivalent Obtained in Example 4

[0114] The expression of β1 integrin in the epidermis equivalentobtained in Example 4 was observed after immunolabelling with the aid ofa mouse monoclonal antibody directed against β1 integrin (Immunotech,Marseille, France, Cat. 1151): After freezing, the skin equivalentsobtained in Example 4 were sectioned into 5 μm thick slices with the aidof a cryostat (make/model). The sections were then rinsed twice with PBSand 25 μl of anti-β1 integrin antibody diluted at 1/50 (Immunotech,Marseille, France, Cat5.1151) were deposited onto each section and leftfor 30 minutes at room temperature (25° C.). The sections were thenrinsed twice with PBS and 25 μl of FITC conjugated antibody (rabbit antimouse FITC, Dako F232) were deposited onto each section and left for 30minutes at room temperature (25° C.). The sections were rinsed twicewith PBS and observed, after mounting, under a LEICA fluorescencemicroscope, model LEITZ DMRB.

[0115] Observation indicated that in the control the β1 integrin wasexpressed in the basal layer of the epidermis reconstructed on thenon-glycated dermis equivalent and in the first suprabasal layer (FIG.1, photo 1), whereas it was expressed in all of the suprabasal layers,up to just under the stratum corneum, in the epidermis reconstructed onthe aged dermis equivalent (FIG. 1, photo 2).

[0116] These results correlate with the observation of a distribution invivo of the expression of the β1 integrin in the basal layer and firstsuprabasal layer in a young subject's skin, and in the basal layer andat least the first 3 suprabasal layers in the skin of an elderlyindividual.

[0117] While the invention has been described in terms of variouspreferred embodiments, the skilled artisan will appreciate that variousmodifications, substitutions, omissions, and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope ofthe-following claims, including equivalents thereof.

What is claimed is:
 1. Aged dermis equivalent, comprising at leastglycated collagen and fibroblasts.
 2. Aged dermis equivalent accordingto claim 1, having a level of glycation of from 2 to
 30. 3. Aged dermisequivalent according to claim 1, having a level of glycation of from 8to
 18. 4. Aged dermis equivalent according to claim 1, wherein theglycated collagen comprises collagen of animal or human origin.
 5. Ageddermis equivalent according to claim 4, wherein the glycated collagencomprises collagen of animal origin.
 6. Aged dermis equivalent accordingto claim 5, wherein the glycated collagen comprises collagen of bovineorigin.
 7. Aged dermis equivalent according to claim 1, wherein theglycated collagen comprises type I collagen.
 8. Aged dermis equivalentaccording to claim 6, wherein the glycated collagen comprises type Icollagen.
 9. Aged dermis equivalent according to claim 1, wherein thefibroblasts comprise fibroblasts of human origin.
 10. Aged dermisequivalent according to claim 5, wherein the fibroblasts comprisefibroblasts of human origin.
 11. Aged dermis equivalent according toclaim 6, wherein the fibroblasts comprise fibroblasts of human origin.12. Aged dermis equivalent according to claim 7, wherein the fibroblastscomprise fibroblasts of human origin.
 13. Epidermis equivalentcomprising at least keratinocytes, said epidermis equivalent beingobtained by seeding of at least keratinocytes onto a dermis equivalentcomprising at least glycated collagen and fibroblasts.
 14. Epidermisequivalent comprising at least keratinocytes, said epidermis equivalenthaving modified expression of β1 integrin.
 15. Epidermis equivalentaccording to claim 14, having expression of β1 integrin in the cells ofat least the first three suprabasal layers.
 16. Epidermis equivalentcomprising at least keratinocytes, said epidermis equivalent havingmodified expression of β1 integrin, said epidermis equivalent beingobtained by seeding of at least keratinocytes onto a dermis equivalentcomprising at least glycated collagen and fibroblasts.
 17. Epidermisequivalent comprising at least keratinocytes, said epidermis equivalenthaving β1 integrin expression in the cells of at least the first threesuprabasal layers, said epidermis equivalent being obtained by seedingof at least keratinocytes onto a dermis equivalent comprising at leastglycated collagen and fibroblasts.
 18. Epidermis equivalent according toclaim 13, wherein the keratinocytes comprise keratinocytes of humanorigin.
 19. Epidermis equivalent according to claim 14, wherein thekeratinocytes comprise keratinocytes of human origin.
 20. Epidermisequivalent according to claim 15, wherein the keratinocytes comprisekeratinocytes of human origin.
 21. Epidermis equivalent according toclaim 16, wherein the keratinocytes comprise keratinocytes of humanorigin.
 22. Epidermis equivalent according to claim 17, wherein thekeratinocytes comprise keratinocytes of human origin.
 23. Epidermisequivalent according to claim 13, further comprising melanocytes and/orLangerhans cells and/or precursors of Langerhans cells.
 24. Epidermisequivalent according to claim 14, further comprising melanocytes and/orLangerhans cells and/or precursors of Langerhans cells.
 25. Epidermisequivalent according to claim 15, further comprising melanocytes and/orLangerhans cells and/or precursors of Langerhans cells.
 26. Epidermisequivalent according to claim 16, further comprising melanocytes and/orLangerhans cells and/or precursors of Langerhans cells.
 27. Epidermisequivalent according to claim 17, further comprising melanocytes and/orLangerhans cells and/or precursors of Langerhans cells.
 28. Epidermisequivalent according to claim 18, further comprising melanocytes and/orLangerhans cells and/or precursors of Langerhans cells.
 29. Aged skinequivalent, comprising at least one epidermis equivalent and one ageddermis equivalent.
 30. Aged skin equivalent according to claim 29,wherein the aged dermis equivalent comprises at least glycated collagenand fibroblasts.
 31. Aged skin equivalent according to claim 29, whereinthe epidermis equivalent is obtained by seeding of at leastkeratinocytes onto a dermis equivalent comprising at least glycatedcollagen and fibroblasts.
 32. Aged skin equivalent according to claim30, wherein the epidermis equivalent is obtained by seeding of at leastkeratinocytes onto a dermis equivalent comprising at least glycatedcollagen and fibroblasts.
 33. Aged skin equivalent according to claim29, wherein the epidermis equivalent comprises at least keratinocytes,said epidermis equivalent having modified expression of β1 integrin. 34.Aged skin equivalent according to claim 33, wherein the epidermisequivalent has expression of β1 integrin in the cells of at least thefirst three suprabasal layers.
 35. Aged skin equivalent according toclaim 29, wherein the epidermis equivalent has modified expression of β1integrin and is obtained by seeding of at least keratinocytes onto adermis equivalent comprising at least glycated collagen and fibroblasts.36. Aged skin equivalent according to claim 29, wherein the epidermisequivalent has β1 integrin expression in the cells of at least the firstthree suprabasal layers, said epidermis equivalent being obtained byseeding of at least keratinocytes onto a dermis equivalent comprising atleast glycated collagen and fibroblasts.
 37. A method for preparing anaged skin equivalent comprising an epidermis equivalent and an ageddermis equivalent which comprises a lattice comprising at least glycatedcollagen and fibroblasts, said method comprising, in a first step,preparing a lattice comprising at least glycated collagen andfibroblasts and, in a second step, reconstructing an epidermisequivalent comprising at least keratinocytes onto the lattice obtainedin the first step.
 38. A method according to claim 37, furthercomprising glycating the collagen used in the first step prior topreparing the lattice.
 39. A method according to claim 38, comprisingglycating the collagen by contacting a solution of at least one collagenand a solution of at least one glycating agent in such manner as toinduce the glycation reaction in vitro in the absence of cells.
 40. Amethod according to claim 37, wherein the collagen comprises collagen ofhuman or animal origin.
 41. A method according to claim 38, wherein thecollagen comprises collagen of human or animal origin.
 42. A methodaccording to claim 39, wherein the collagen comprises collagen of humanor animal origin.
 43. A method according to claim 40, wherein thecollagen comprises collagen of animal origin.
 44. A method according toclaim 41, wherein the collagen comprises collagen of animal origin. 45.A method according to claim 42, wherein the collagen comprises collagenof animal origin.
 46. A method according to claim 43, wherein thecollagen comprises collagen of bovine origin.
 47. A method according toclaim 44, wherein the collagen comprises collagen of bovine origin. 48.A method according to claim 45, wherein the collagen comprises collagenof bovine origin.
 49. A method according to claim 37, wherein thecollagen comprises type I, III or V collagen.
 50. A method according toclaim 40, wherein the collagen comprises type I, III or V collagen. 51.A method according to claim 46, wherein the collagen comprises type I,III or V collagen.
 52. A method according to claim 49, wherein thecollagen comprises type I collagen.
 53. A method according to claim 50,wherein the collagen comprises type I collagen.
 54. A method accordingto claim 51, wherein the collagen comprises type I collagen.
 55. Amethod according to claim 52, wherein the collagen comprises bovine typeI collagen.
 56. A method according to claim 53, wherein the collagencomprises bovine type I collagen.
 57. A method according to claim 54,wherein the collagen comprises bovine type I collagen.
 58. A methodaccording to claim 37, wherein the collagen is at a concentration offrom 2 mg/ml to 6 mg/ml.
 59. A method according to claim 58, wherein thecollagen is at a concentration of from 3 mg/ml to 5 mg/ml.
 60. A methodaccording to claim 58, wherein the collagen is bovine type I collagen.61. A method according to claim 59, wherein the collagen is bovine typeI collagen.
 62. A method according to claim 39, wherein the glycatingagent is an agent which is capable of reacting with an amino group ofthe collagen to form a Schiff's base according to the Maillard reaction.63. A method according to claim 62, wherein the glycating agentcomprises glucosone, 3-deoxyglucosone, glyoxal, methylglyoxal or asugar.
 64. A method according to claim 63, wherein the glycating agentis a sugar.
 65. A method according to claim 64, wherein the sugarcomprises an ose.
 66. A method according to claim 65, wherein the osecomprises ribose, fructose or glucose.
 67. A method according to claim39, wherein the glycating agent comprises ribose or glucose.
 68. Amethod according to claim 39, wherein the amount of glycating agent isfrom 0.5% to 20% by weight of the total weight of the collagen solution.69. A method according to claim 68, wherein the amount of glycatingagent is from 1% to 10% by weight of the total weight of the collagensolution.
 70. A method according to claim 38, wherein the glycationreaction is carried out at a temperature of from 15° C. to 30° C.
 71. Amethod according to claim 39, wherein the glycation reaction is carriedout at a temperature of from 15° C. to 30° C.
 72. A method according toclaim 70, wherein the glycation reaction is carried out at a temperatureof from 20° C. to 25° C.
 73. A method according to claim 38, wherein theduration of the glycation reaction is from 15 days to 2 months.
 74. Amethod according to claim 39, wherein the duration of the glycationreaction is from 15 days to 2 months.
 75. A method according to claim73, wherein the duration of the glycation reaction is from 25 days to 35days.
 76. A method according to claim 74, wherein the duration of theglycation reaction is from 25 days to 35 days.
 77. A method according toclaim 37, wherein the collagen is a mixture of preglycated collagen andnon-glycated collagen.
 78. A method according to claim 77, wherein theratio of glycated collagen to non-glycated collagen is from 25 to 75.79. A method according to claim 78, wherein the ratio of glycatedcollagen to non-glycated collagen is from 45 to
 55. 80. A methodaccording to claim 37, wherein the keratinocytes comprise keratinocytesof human origin.
 81. A method according to claim 38, wherein thekeratinocytes comprise keratinocytes of human origin.
 82. A methodaccording to claim 39, wherein the keratinocytes comprise keratinocytesof human origin.
 83. Use of an aged dermis equivalent as claimed inclaim 1, in the preparation of epidermis and/or aged skin equivalent.84. Use of an aged dermis equivalent as claimed in claim 1, in the studyof the glycation phenomenon and modulators of said phenomenon, in thestudy of photoaging and the effects of ultraviolet rays on the dermisand modulators of said effects or in the study of the influence ofglycation on the components of the dermis.
 85. Use of an aged epidermisequivalent as claimed in claim 13, in the study of the glycationphenomenon and modulators of said phenomenon, in the study of phenomenalinked to aged epidermis, in the study of modulators of the appearanceof wrinkles, in the study of photoaging and the effect of ultravioletrays on the epidermis and modulators of said effect or in the study ofthe influence of glycation on the components of the epidermis and theannexes of the skin.
 86. Use of an aged skin equivalent as claimed inclaim 29, in the study of the glycation phenomenon and modulators ofsaid phenomenon, in the study of phenomena linked to aged skin and/orepidermis, in the study of modulators of the appearance of wrinkles, inthe study of photoaging and the effect of ultraviolet rays on the skinand/or the dermis and/or the epidermis and modulators of said effect, inthe study of the influence of glycation on the components and/or annexesof the skin and/or dermis and/or epidermis, or in the study of thecomplications caused by diabetes via glycation.